The present invention relates to a stable isotope-labeled amino acid and method for incorporating the same into target protein, and NMR method for the structural analysis of protein. The present invention also relates to a method for producing regio-selectively stable isotope-labeled fumaric acid and tartaric acid. In particular, the present invention relates to a method for efficiently producing symmetric and asymmetric stable isotope-labeled fumaric acid and also a method for producing stable isotope-labeled tartaric acid with a high optical purity.
In the determination of the protein structure by NMR, a sample uniformly labeled with stable isotopes such as 13C/15N has so far been used. However, this technique sharply becomes difficult when the molecular weight of the protein exceeds 20,000. There were proposed methods for solving this problem such as a method wherein about 50 to 80% of hydrogen in the protein is replaced at random with deuterium (2H) in addition to the labeling with 13C/15N to utilize NMR signals of the nucleus of remaining hydrogen (1H) and a method wherein all the hydrogen atoms except those in methyl group and aromatic ring in the amino acid residue are replaced with deuterium. However, the subject and the utility of those conventional methods are limited because these techniques sacrificed of the accuracy of structural information to determine the structure of high-molecular weight proteins.
[1-13C, 2,3-2H2]phenylalanine, [2H2]serine and [2H2]alanine are described in a paragraph of Analysis of higher-order structure of protein by main chain carbonyl 13C-NMR of Protein III Higher-order structure in “Shin Seikagaku Jikken Koza (Lectures on New Biochemistry Experiments)” I published by Tokyo Kagaku Dojin on Nov. 15, 1990. However, this technique is employed for the determination of dihedral angle χ of amino acids by a specific multi-labeling method. It has not yet been tried that not only the labeled amino acids but also other amino acids are labeled and these amino acids are incorporated into a target protein to analyze the stereostructure thereof.
On the other hand, in the analysis of biological organic compounds such as nucleic acids and protein by NMR or mass spectra, fumaric acid and tartaric acid labeled with stable isotopes such as 13C and 2H are widely used. Recently, the following technique for analyzing the structure of protein is employed: isotope-labeled amino acids are derived from isotope-labeled fumaric acid and tartaric acid and the structure of protein is analyzed by using those amino acids by NMR. Under those circumstances, the demand of stable isotope-labeled fumaric acid and also isotope-labeled tartaric acid is expected to increase.
However, in fact, the stable isotope-labeled fumaric acid is quite expensive (for example, 0.1 g of 1,2,3,4-13C4 fumaric acid costs at least 100,000 yen) and stable isotope-labeled tartaric acid is not available on the market.
Various methods for synthesizing isotope-labeled fumaric acid were so far reported. For example, there are known a method wherein a malonic ester is synthesized (E. C. Jorgensen et al., J. Am. Chem. Soc., 74, 2418, 1952), a method wherein a reaction for leaving dibromosuccinic acid is employed (R. F. Nystrom et al., J. Am. Chem. Soc., 74, 3434, 1952) and a method wherein the reduction of acetylenedicarboxylic acid is employed in the course of the reactions (Y. J. Topper et al., J. Biol. Chem. 177, 303, 1949).
For synthesizing stable isotope-labeled tartaric acid, for example, dihydroxylation of fumaric acid by the oxidation with osmium is known (H. Erlenmeyer et al., Helv. Chim. Acta, 22, 701, 1939; and E. C. Jorgensen et al., J. Am. Chem. Soc., 74, 2418, 1952).
However, those known methods have problems that they are unsuitable for the large-scale production of stable isotope-labeled fumaric acid or tartaric acid and that according to those methods is difficult to control the regio-selectivity. For example, methods of E. C. Jorgensen et al. and R. F Nystrom et al. for synthesizing stable isotope-labeled fumaric acid are both suitable for the synthesis in a small scale, but the increase in the scale is difficult. Further, a method of Y. J. Topper is unsuitable for the synthesis of asymmetric fumaric acid such as 1-13C fumaric acid or 2-13C fumaric acid while it is suitable for the synthesis of labeled symmetric fumaric acid such as 1,4-13C2 fumaric acid, 2,3-13C2 fumaric acid or 1,2,3,4-13C4 fumaric acid.
In the method of H. Erlenmeyer et al. for synthesizing labeled tartaric acid, the obtained tartaric acid is racemic and, therefore, the optical resolution is necessary for isolating L-tartaric acid or D-tartaric acid. This method cannot be easily accepted as a useful method wherein the expensive, stable isotope is used because the yield of the product is reduced by the optical resolution.